Assay Development for the Identification of NEDD8- activating Enzyme Inhibitors

鉴定 NEDD8 激活酶抑制剂的检测方法开发

• 批准号: 8977494
• 负责人: Matthew Petroski
• 金额: $ 44.61万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-03 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8977494
• 关键词: Affinity; Basic Science; Binding; Biochemical; Biological; Biological Assay; Cancer Patient; Cell physiology; Cells; Chemicals; Chronic; Clinical; Clinical Data; Clinical Trials; Cullin Proteins; Data; Development; Disease; Enzyme Inhibition; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Kinetics; Enzymes; Equilibrium; Etiology; Experimental Neoplasms; Health; Human; Inflammation; Libraries; Ligands; Lysine; Malignant Neoplasms; Measures; Mediating; Mutation; Normal Cell; Pharmaceutical Preparations; Phase; Post-Translational Protein Processing; Property; Proteins; Regulation; Research; Resistance; Role; Signal Pathway; Specificity; System; Testing; Therapeutic; Therapeutic Agents; Translational Research; Ubiquitin; Ubiquitin Like Proteins; Work; adenylate; antitumor effect; assay development; base; cancer cell; cancer therapy; enzyme activity; high throughput screening; improved; in vivo; inhibitor/antagonist; innovation; insight; interest; mimetics; multicatalytic endopeptidase complex; novel; novel therapeutic intervention; pre-clinical; preclinical study; response; screening; small molecule; small molecule inhibitor; small molecule libraries; tool; ubiquitin ligase

DESCRIPTION (provided by applicant): The ubiquitin-like protein NEDD8 functions to regulate the activation of cullin-RING ubiquitin ligases (CRLs). Although CRLs have well-established roles in many fundamental aspects of cellular physiology, recent pre- clinical studies have found that cancer cells are more reliant on NEDD8 for proliferation and survival than normal cells. These observations have invigorated interest in devising strategies to pharmacologically inhibit NEDD8 to treat cancer and other diseases. The NEDD8-activating enzyme (NAE) represents the most attractive target of the NEDD8 system as it possess a catalytic pocket suitable for binding small molecules and undergoes structural rearrangements important for its activities. MLN4924, a mechanism-based inhibitor that generates a NEDD8-adenylate mimetic in the NAE catalytic pocket, represents the only NEDD8 system inhibitor and is currently being evaluated on cancer patients. We and other have found that cancer cells and experimental tumors rapidly lose sensitivity to MLN4924 through treatment-emergent mutations in NAE that impact inhibitor binding by altering the enzyme's biochemical properties. Although the implications of these on MLN4924 therapy are currently unknown, they suggest cancer cells tolerate considerable variability in NAE function. These mutations render cells broadly resistant to molecules targeting the enzyme's catalytic pocket. This project, submitted in response to PAR-13-364 Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery, seeks to develop highly innovative biophysical and cell-based assays focused on the NEDD8 system and to use these in pilot screens to identify proof-of-concept probes. Through our preliminary studies, we have devised a homogeneous biophysical assay that measures ligand- dependent changes in NAE thermal stability. We hypothesize that this assay will allow for different classes of equilibrium-binding reversible inhibitors to be identifie by screening chemically diverse small molecule libraries. To test this hypothesis and achieve the overall objective of the research, three Specific Aims are proposed: 1) to establish and validate biophysical assays that measure NAE thermal stability and supporting specificity assays; 2) to develop cell-based assays that measure biological effects of NAE inhibition; and 3) to conduct focused high-throughput screens with selected libraries, verify hits, and determine hit potency and selectivity. IMPACT: This work is highly innovative with translational relevance based on the extraordinary opportunity it provides to develop new research tools and therapeutic approaches to inhibit NAE and the NEDD8 system for the treatment of cancers and other diseases. Through our efforts focused on generating novel assays and chemical probes, these studies are expected to provide important new insight into the NEDD8 system and other ubiquitin and ubiquitin-like protein modification systems to improve human health.

描述（由申请人提供）：泛素样蛋白NEDD 8的功能是调节cullin-RING泛素连接酶（CRL）的激活。尽管CRL在细胞生理学的许多基本方面具有公认的作用，但最近的临床前研究发现，癌细胞比正常细胞更依赖于NEDD 8来增殖和存活。这些观察结果引起了人们对设计抑制NEDD 8以治疗癌症和其他疾病的策略的兴趣。NEDD 8激活酶（NAE）是NEDD 8系统最具吸引力的靶标，因为它具有适合结合小分子的催化口袋，并经历对其活性重要的结构重排。MLN 4924是一种基于机制的抑制剂，在NAE催化口袋中产生NEDD 8-腺苷酸模拟物，代表唯一的NEDD 8系统抑制剂，目前正在癌症患者中进行评价。我们和其他研究人员发现，癌细胞和实验性肿瘤通过NAE中治疗后出现的突变迅速丧失对MLN 4924的敏感性，这些突变通过改变酶的生化特性影响抑制剂结合。尽管这些对MLN 4924治疗的影响目前尚不清楚，但它们表明癌细胞耐受NAE功能的相当大的变异性。这些突变使细胞对靶向酶催化口袋的分子具有广泛的抵抗力。该项目是为了响应PAR-13-364“开发用于探针和治疗前发现的高通量筛选测定法”而提交的，旨在开发高度创新的生物物理和基于细胞的测定法，重点关注NEDD 8系统，并在中试筛选中使用这些方法来识别概念验证探针。通过我们的初步研究，我们设计了一种均相生物物理测定法，其测量NAE热稳定性的配体依赖性变化。我们假设，该测定将允许通过筛选化学上多样化的小分子文库来鉴定不同类别的平衡结合可逆抑制剂。为了验证这一假设并实现研究的总体目标，提出了三个具体目标：1）建立和验证测量NAE热稳定性的生物物理测定法和支持特异性测定法; 2）开发测量NAE抑制的生物学效应的基于细胞的测定法; 3）用选定的文库进行集中的高通量筛选，验证命中，并确定命中效力和选择性。影响：这项工作具有高度创新性，具有翻译相关性，因为它提供了开发新的研究工具和治疗方法的非凡机会，以抑制NAE和NEDD 8系统用于治疗癌症和其他疾病。通过我们专注于产生新的检测方法和化学探针的努力，这些研究有望为NEDD 8系统和其他泛素和泛素样蛋白修饰系统提供重要的新见解，以改善人类健康。